The identification of transmitter substances at specific neural sites is a problem of basic importance for the Neurosciences. Proposed studies are designed to detect neurotransmitters released by nerve stimulation from specific synaptic sites in isolated preparations of spinal cord and retina. Ionic and pharmacological manipulations will be employed to determine which transmitters may be released from which synaptic sites. Metabolic compartmentation of the amino acids glutamate and glycine will be examined in discrete regions of spinal cord where these compounds have been proposed to function as transmitters. The effects of orthodromic nerve stimulation on these metabolic patterns will also be studied. By this approach the mechanisms may be determined by which a common intermediate metabolite may also function in the highly specific manner required of a neurotransmitter. These studies may help to define a biochemical criterion of transmitter identification that will be useful for examining these amino acids as transmitters at oter synaptic sites. Amino compounds released during nerve stimulation will be assayed in tissue perfusion media by quantitative estimation of their respective derivatives formed with H3-dinitrofluorobenzene (DNFB). This procedure, involving separation of the derivatives by thin-layer microelectrophoresis and chromatography, has a working sensitivity of 30-60 pmoles of individual amino compounds. The method is also applicable to determinations of specific radioactivities (SRA) of tissue metabolites following incubation with C14-precursors. SRA is determined in a single-step procedure comprising double isotope counting of each H3-dinitrophenyl-C14-amino compound derivative.